Pe anti pdl 1 clone 10f 9g2

Cells were stained with a combination of surface marker antibodies detailed below. Primary direct staining was performed with antibodies purchased from Biolegend: FITC anti-CD86, clone RMMP-2; FITC anti-MHC-II (I-A/I-E), clone M5/114.15.2; FITC anti-CD25, clone 3C7; PE anti-MHC-I, clone AF6-88.5; PE Biotin anti-CD80, clone 1610A1; PE anti-PDL1, clone 10F.9G2; PE anti-CD137, clone 17B5; APC/Cy7 anti-CD69, clone H1.2F3. For indirect staining antibodies were purchased from Biolegend: Biotin anti-IFN-γ, clone XMG1.2; from eBioscience: Biotin anti-SIINFEKL/H2-K^b, clone 25-D1.16. Here, applicable cells were stained with secondary Streptavidin-FITC (Invitrogen) or FITC anti-rat IgG, clone Poly4054 (Biolegend). Staining was carried out for 20–30 min at 4°C in FACS buffer containing 0.5% sodium azide in PBS. Samples were acquired using the Epics XL-MCL flow cytometer (Beckman Coulter, Miami, FL, USA) and analyzed using FlowJo software. Fold change in MFI was calculated by using the following formula: fold change = [(SIINFEKL MFI − Negative MFI)/Negative MFI], where “SIINFEKL” refers to cells that were pulsed with SIINFEKL peptide and “Negative” refers to unpulsed cells.